Immunorhelin compounds for intracellular infections

ABSTRACT

The present invention provides immune stimulating peptides (immunorhelins) capable of activating GnRH receptors when administered to animal or human patients or cells. These immunorhelins have utility in treating intracellular bacterial, fungal, and protozoal infections.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format via USPTO's Patent Center and ishereby incorporated by reference in its entirety. Said XML copy, createdon May 16, 2023, is named 54397US_P3376USPC01_Sequence_Listing.xml andis 98,927 bytes in size.

FIELD OF THE INVENTION

The present invention provides a set of novel peptides capable ofstimulating the immune system, named immunorhelins. The invention alsoprovides novel peptides capable of stimulating GnRH receptors onleukocytes. The present invention relates to novel compounds as such andto the compounds for use in medicine, notably in the treatment ofintracellular infections. The immunorhelins may also be used asimmunomodulating adjuvants in vaccination. The novel GnRH receptorstimulating immunorhelins maximizes the modulating effects of the immunesystem while minimizing the therapeutically unwanted endocrine effects.The present invention also provides methods for preparing immunorhelinsof the invention that have improved properties for use in medicine.

BACKGROUND OF THE INVENTION

Intracellular bacterial, fungal, and protozoal infections are often notdiagnosed in healthy individuals as they appear asymptomatic, or becausethe symptoms are mild enough that the infected individual is notinclined to seek medical assistance. As such, intracellular infectionsmay persist latently or may progress to a disease state. Conditionsinterfering with normal T cell function usually leads to progression ofthe disease from a latent infection and intracellular infections such asMycobacterium tuberculosis (Mtb) are a common cause of death in patientswhere HIV infection has progressed to AIDS. There is thus also a greatneed in the art for methods and means of treating infections.

Intracellular pathogens such as Mtb have the capacity to hide withinintracellular compartments in monocytes and macrophages causingpersistent infections. Although Mtb are recognized by CD4⁺ T helpercells in the lung and an appropriate response is mounted, the systemfails to create sterilizing immunity (MacMicking 2012).To escape immunerecognition by the host, Mtb have developed a series of mechanism thatinhibits recognition of Mtb peptides presented in the MHC class IIpocket for CD4⁺ T helper cells. Toll like receptor 2 has beendemonstrated to be inhibited by Mtb, which in turn inhibits IFN-γinduced MHC class II expression (Noss 2001). In addition, data suggestthat Mtb has the capacity to inhibit phagosome processing andmaturation, possibly by an invariant chain associated mechanism(Ramachandra 2001). Therefore, the normal antigen processing, loadingand presentation of MHC class II peptides derived from Mtb is impaireddue to Mtb produced immune escape factors.

The endosomal lysosomal pathway is designed to take up pathogens,process them into 12-15 aa long peptides, peptides, that after theremoval of the Invariant chain peptide CLIP by HLA-DM, are loaded intothe MHC class II pocket. The antigen loading is followed by transport ofthe MHC class Il-peptide complex to the cell surface for presentationfor the specific T cell receptor of CD4⁺ T helper cells (Roche 2015).Recently the Mtb expressed protein EsxH has been reported to directlyinhibit the endosomal sorting complex required for transport (ESCRT)machinery (Portal-Celhay 2016). EsxH inhibits the ability of antigenpresenting monocytes and macrophages to activate CD4⁺ T helper cells.Since intact ESCRT machinery seems necessary for antigen processing,presentation and activation of T cells, EsxH is the link that explainsMtb induced immune escape by intervening with the MHC class II pathway.

The importance of MHC class II presentation has also been demonstratedin patients with primary immunodeficiencies (PID). PID patients withdefects in the IFN-γ circuit, involving IFNGR, IL-12 have an increasedof acquiring TBC and atypical mycobacterial infections. Since MHC classII expression is dependent and regulated by IFN-γ expression defects inthe IFN-γ circuit will result in additionally decreased MHC class IIexpression and a poor activation of CD4⁺ T helper cells.

Protozoa such as Toxoplasma gondii have developed a mechanism to avoidimmune recognition by hiding intracellularly as an obligateintracellular parasite. The mechanism involves interference with MHCclass II expression and thus diminish the amount of Toxoplasma gondii tobe presented for specific CD4⁺ T helper cells. The detailed mechanism isdependent on soluble proteins expressed by Toxoplasma gondii thatinhibit IFN-gamma induced expression of MHC class II (Leroux 2015).

Furthermore, it has been demonstrated that different fungal infectionsare dependent on MHC class II expression. Cryptococos neoformans maycause life threatening brain infections in patients withimmunodeficiencies including HIV. Work in a mouse model of Cryptococosneoformans has demonstrated that the activation of microglial cells andtheir upregulation of MHC class II, in an IFN-gamma dependent manner, iscritical for survival (Zhou 2007).

Therefore, to overcome the immune escape mechanisms induced by Mtb andother intracellular bacteria, protozoa such as Toxoplasma gondii, orfungi exemplified by Cryptococus an increased expression of MHC class IIand MHC class I on the cell surface of monocytes, macrophages, microgliaor other infected cells is likely beneficial for immune recognition andelimination of the pathogen

INTRODUCTION TO THE INVENTION

GnRH I (also known as gonadotropin releasing hormone or LHRH), is adecapeptide with the structurepyroGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂. It is produced as a 92amino-acid propeptide which is modified post-translationally to form thefinal peptide with pyroglutamic acid at the amino terminus and acarboxamide at the carboxyl terminus. It has long been known that it isresponsible for release of FSH and LH from the anterior pituitary gland,and is normally released from the hypothalamus in a pulsative manner.Supraphysiological levels of GnRH I induce an immediate increase of FSHand LH secretion, soon followed by inhibition of FSH and LH secretion.This is due to the fact that high levels of GnRH I have an inhibitoryeffect on the type I GnRH receptors of the anterior pituary gland.Continuous administration of GnRH I at high unphysiological levels thusinduces pharmacological castration (Fink 1998). A large number of GnRH Iagonists and antagonists have been synthesized for use in therapeuticareas such as hormone sensitive cancer. Initially, salts of GnRH I wereused therapeutically (such as gonadorelin hydrochloride and gonadorelindiacetate tetrahydrate). Further drug discovery and development led tothe clinical use of a wide variety of agents, including buserelin,triptorelin, nafarelin, histrelin and leuprorelin, each of which hasimprovements over gonadorelin such as extended half-life andsuper-agonism of the type I GnRH receptor.

It has been reported that GnRH I not only exhibits hormonal effects butalso may stimulate the immune system (Jacobson and Ansari 2004). McCleanand McCluggage (McClean and McCluggage 2003) observed massiveinfiltration of small mature lymphocytes in uterine leiomyomas afterpreoperative treatment with a type I GnRH receptor agonist. Bardsley etal (Bardsley 1998) made the same observation, indicating a stimulatoryeffect on migration of GnRH I on the immune cells. Reports have beenmade on chronic plasma cell endometritis in hysterectomy specimens fromHIV-infected women in a retrospective analysis (Kerr-Layton 1998), andon elevated levels of FSH and LH (hypergonadotropic) in HIV-infected men(Arver 1999 and Brockmeyer 2000). By administering GnRH Itodiabetes-prone BB rats exhibiting an AIDS-like lym-phocyte profile theCD4 T-lymphocyte numbers was increased (Jacobson 1999).

WO 2009/145690 A1 teaches us that GnRHs activate and upregulate MHCclass I on T cells with the notion that HIV infected CD4 T cells downregulate MHC class I due to the HIV encoded protein Nef thereforeavoiding recognition (Lubben et al., 2007

In humans, two variants of GnRH peptide exist, GnRH I and GnRH II, codedfor by dif-ferent genes. The structure of GnRH II ispyroGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-GlyNH₂ (differences from GnRH Iunderlined). GnRH II is a nonhypothalamic form primarily producedoutside the brain, and has been suggested to be involved in thenon-endocrine aspects of the GnRH system (White 1998). Surprisingly, wefound an effect of GnRH II stimulation on the MHC class I expression onT cells demonstrating that GnRH II directly activates these cells (FIG.1 ).

Unlike other mammals, only one conventional human GnRH receptor has beendescribed, the type I GnRH receptor. The type II GnRH receptor homologueis present on chromosome 1q12 gene in humans but contains a frame shiftand a stop codon and is believed not to be functionally expressed(Morgan 2003). Surprisingly, our findings suggest that the type II GnRHreceptor is indeed expressed on T cells as they respond to GnRHstimulation by increased MHC class I expression (FIG. 1 ). Thesefunctional findings were substantiated by qPCR analysis where we coulddemonstrate expression of the type II GnRH receptor mRNA. In addition,the relative expression level of the type II GnRH receptor was highercompared to the expression levels of the type I GnRH receptor on naiveand memory T cells (FIG. 4 ). Thus, we have identified that theexpression of the type II GnRH receptor is the dominant receptorexpressed on T cells, functionally responsive to GnRH stimulus.

We have also discovered that GnRH I analogs may activate T cells leadingto MHC class I expression. In a recent clinical trial using the GnRH Ianalog Buserelin as treatment for HIV, HIV infected men were providedwith sex hormone substitution to minimize the endocrine effects of GnRHI. These effects are mediated by GnRH I binding to pituitary type I GnRHreceptors, causing decreased testosterone production and subsequentlyimpotence. It is very likely that GnRH I in addition to its endocrineeffects cross-signal and stimulate the immune system by binding to thetype II GnRH receptor on T cells when high castrating levels of GnRHanalogues are used. Interestingly, GnRH I binding to receptors expressedin breast cancer cells displays a low binding affinity (Kd,1.6-3.0×10(−6) M), whereas central pituitary binding of GnRH I displaysa 1000-fold higher affinity (Kd, 4.8×10(−9) M) (Eidne 1987).

It is likely that the difference in binding affinity of GnRH I and GnRHII peptides reflects the expression of type I GnRH receptors specialisedfor GnRH I binding on pituitary cells, whereas peripheral cells may havedominated expression of type II GnRH receptor and therefore low affinityand an “off target” effect of GnRH I binding. Thus, our unexpectedfinding that the type II GnRH receptor is the dominating receptor on Tcells is novel and may explain the receptor physiology of GnRH land GnRHII. Therefore, by using GnRH II-like peptides in the treatment of HIVthe endocrine effect should be minimized and the immune stimulationeffect isolated and enhanced.

DESCRIPTION OF THE INVENTION

Based on these discoveries, the inventors have made GnRH II-likepeptides, termed immunorhelins, in order to optimise immune stimulatingeffects and minimize the effect on the hormonal system. Theseimmunorhelins have use in stimulating MHC class I capable of leading toan immune response clearing infectious agents, such as intracellularinfections, and in treating or co-treating HIV or cancer. Therefore,several GnRH II-like peptides are disclosed which have potent binding totype II GnRH receptors but preferably weaker binding to type I GnRHreceptors, leading to a comparable or stronger MHC class I response, buta weaker ‘off-target’ effect on hormone stimulation or inhibition. Inthe case that GnRH II-like peptides of the invention also bind andactivate Type I GnRH receptors thereby stimulating endocrine signalling,the compounds of the present invention may therefore be administeredtogether with one or more natural, semi-synthetic or synthetic sexhormones to counter the endocrine effects of GnRH II-like peptides, e.g.testosterone or oestrogen depending on the hormonal status of thepatient. In an adult male person the natural, semi-synthetic orsynthetic sex hormone is testosterone or an agent having a correspondinghormonal effect. In an adult female person the natural, semi-syntheticor synthetic sex hormone is oestradiol or an agent having acorresponding hormonal effect, in particular in combination with aprogestogen. The latter is added to avoid the development of endometrialcancer in the female and to avoid vaginal bleedings. Hysterectomizedwomen, however, do not benefit from the addition of progestogen.

Viral peptides emerge in the cytosol and are targeted to the proteosomefollowed by processing and transport (TAP1 and TAP2) into theendoplasmatic reticulum where digested HIV peptides are added into theMHC class I peptide, followed by transport to the cell surface forpresentation.

In contrast to the teaching of WO 2009/145690 A1 and in known art,intracellular bacteria are residents of the endosomal lysosmal pathway,a pathway normally not accessible for MHC class I in normal cells suchas in T cells. However, in the present invention we demonstrate for thefirst time that GnRHs can upregulate MHC class I on antigen presentingcells (APCs) such as CD14+ monocytes (FIG. 2 ). APCs have the uniquecapability of cross presentation, i.e. they can present antigens fromthe endosomal lysosmal pathway into the MHC class I pocket, foractivation of CD8+ T cells. This finding was surprising and suggeststhat intracellular bacteria such as M Tuberculosis which reside andhides in the endosomal lysosomal pathway of APCs. Thus, treating MTuberculosis infected individuals with GnRHs will activate and stimulateAPCs to initiate MHC class I upregulation by the cross-presentationpathway leading to recognition of M Tuberculosis peptides by CD8+ Tcells and elimination of infected APCS.

The present invention also provides a method for treating intracellularinfections such as infections by bacteria, protozoa and fungi by use ofone or more GnRH analogs. Intracellular bacteria to be treated includeMycobacterium tuberculosis, Mycobacteria causing atypical disease,Mycobacterium avium and M. intracellulare (also known as Mycobacteriumavium-intracellulare complex, or MAC), M. kansasii, M. marinum, M.fortuitum, M. gordinae, Mycoplasma pneumoniae, M. genitalium, M.hominis, Ureaplasma urealyticum, U. parvum, Chlamydophila pneumoniae,and Salmonella typhimurium. Intracellular protozoa include Toxoplasmagondii, Plasmodium falciparum, P. vivax, Trypanosoma cruzi,Cryptosporidium, and Leishmania. Intracellular fungi include Histoplasmacapsulatum, Cryptococcus neoformans, and Encephalitozoon cuniculi.

The present invention provides peptide-based analogues of human GnRH II.

The compounds of the invention is contemplated to induce improved MHC IIand/or MHC I antigen presentation, which make them useful in thetreatment of intracellular bacterial, fungal, and protozoal infections.The present invention further provides a method for treatingintracellular infection which comprises administration of anunphysiological amount of GnRH or a GnRH analog, and preferably alsoadministering a sex hormone

Thus, in one aspect of the invention there is provided a peptide ofFormula (I):

or a pharmaceutically acceptable salt thereof, and wherein

R₁=

R₂=

R₃=

R₄=

R₅═Me, Et, CH₂CF₃, iPr, nPr, nBu, iBu, sBu, tBu, cyclopropyl, CH₂CONH₂,or NHCONH₂

Formula I regarding the compounds of the invention can also be expressedas: pGlu-His-Trp-Ser-AA₁-AA₂-AA₃-AA₄-Pro-X,

wherein:

AA₁ is selected from His and Tyr

AA₂ is selected from D-Ser(OtBu), D-Trp, D-Nal, D-Bhi, and D-Leu

AA₃ is selected from Leu and Trp

AA₄ is selected from Arg and Tyr

X is selected from —NHMe, —NHEt, —NHCH₂CF₃, —NHiPr, —NHnPr, —NHnBu,—NHiBu, —NHsBu, —NHtBu, —NHcyclopropyl, —NH—NH—CONH₂ and —NHCH₂CONH₂

The invention does not include the following compounds of formula (I):

Pro- viso R1 R2 R3 R4 R5 P1

CH₂CONH₂ P2

CH₂CONH₂ P3

CH₂CONH₂ P4

CH₂CONH₂ P5

CH₂CONH₂ P6

CH₂CONH₂ P7

Et P8

Et P9

CH₂CONH₂ P10

CH₂CONH₂ P11

CH₂CONH₂ P12

Et P13

Et P14

Et P15

NHCONH₂ P16

NHCONH₂ P17

Et P18

NHCONH₂ P19

Et P20

NHCONH₂ P21

Et

Disclaimed are compounds of formula (I) expressed as:pGlu-His-Trp-Ser-AA₁-AA₂-AA₃-AA₄-Pro-X, where AA₁ is His, AA₃ is Trp,AA₄ is Tyr and AA₂ is selected from D-Leu, D-tBu-Ser and D-Trp, and X is—NHEt or NH—NH—CONH2.

The compounds P1-P15 excluded from the invention can also be expressedas follows:

P1. pGlu-His-Trp-Ser-His-D-Ser(tBu)-Trp-Tyr-Pro-Gly-NH₂

P2. pGlu-His-Trp-Ser-His-D-Trp-Leu-Arg-Pro-Gly-NH₂

P3. pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Arg-Pro-Gly-NH₂

P4. pGlu-His-Trp-Ser-His-D-Trp-Trp-Arg-Pro-Gly-NH₂

P5. pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-Gly-NH₂

P6. pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-Gly-NH₂

P7. pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-NHEt

P8. pGlu-His-Trp-Ser-His-D-Nal-Trp-Tyr-Pro-NHEt

P9. pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-Gly-NH₂

P10. pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂

P11. pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-Gly-NH₂

P12. pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHEt

P13. pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Arg-Pro-NHEt

P14. pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Arg-Pro-NHEt

P15. pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Arg-Pro-NHNHCONH₂

An interesting selection of compounds of the invention is compounds ofFormula (I), or a pharmaceutically acceptable salt thereof, and wherein:

Group Type I Type II R₁

R₃

R₄

and wherein at least one of R₁, R₃ and R₄ are selected from type II, andthose of R₁, R₃ and R₄ which are not selected from type II, are selectedfrom Type I,

wherein R₂ 32

and wherein R₅═Me, Et, CH₂CF₃, iPr, nPr, nBu, iBu, sBu, tBu, cyclopropylor, CH₂CONH₂

The selection of compounds can also be expressed as:

pGlu-His-Trp-Ser-AA₁-AA₂-AA₃-AA₄-Pro-X,

wherein AA₁, AA₂, AA₃, AA₄ and X are as defined above and wherein atleast one of AA₁, AA₃ and AA₄ is selected from His, Trp and Tyr; theremaining AA₁, AA₃ and AA₄ is selected from Tyr, Leu, Arg and withexclusion of the compounds P1-P15 as specified above and those othercompounds disclaimed in claim 1.

In an embodiment, two or three of R₁, R₃ and R₄ are selected from typeII according to the list above, and the remaining R₁, R₃ and R₄ areselected from type I.

In an embodiment one of R₁, R₃ and R₄ is selected from Type I accordingto the list above and two of R₁, R₃ and R₄ are selected from type IIaccording to the list above.

and with exclusion of the compounds P1-P21 as specified above.

Specific compounds according to the invention include:

Com- pound no. R1 R2 R3 R4 R5  1

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-Gly-amide,  2

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-Gly-amide  3

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-Gly-amide  4

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Ser(tBu)-Trp-Arg-Pro-Gly-amide  5

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Tyr-Pro-Gly-amide  6

Et pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-NHEt  7

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Tyr-Pro-Gly-amide  8

Et pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-NHEt  9

Et pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-NHEt 10

Et pGlu-His-Trp-Ser-His-D-Ser(tBu)-Trp-Arg-Pro-NHEt 11

Et pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Tyr-Pro-NHEt 12

Et pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Tyr-Pro-NHEt 13

Et pGlu-His-Trp-Ser-His-D-Ser(tBu)-Trp-Tyr-Pro-NHEt 14

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Tyr-Pro-Gly-amide 15

Et pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-NHEt 16

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Trp-Leu-Tyr-Pro-Gly-amide 17

Et pGlu-His-Trp-Ser-His-D-Trp-Leu-Arg-Pro-NHEt 18

Et pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Arg-Pro-NHEt 19

Et pGlu-His-Trp-Ser-Tyr-D-Trp-Leu-Tyr-Pro-NHEt 20

Et pGlu-His-Trp-Ser-His-D-Trp-Trp-Arg-Pro-NHEt 21

Et pGlu-His-Trp-Ser-His-D-Trp-Leu-Tyr-Pro-NHEt 22

Et pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-NHEt 23

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-Gly-amide 24

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-Gly-amide 25

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-Gly-amide 26

Et pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-NHEt 27

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Nal-Trp-Arg-Pro-Gly-amide 28

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Nal-Leu-Tyr-Pro-Gly-amide 29

Et pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-NHEt, 30

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Tyr-Pro-Gly-amide 31

Et pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-NHEt 32

Et pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-NHEt 33

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Nal-Trp-Tyr-Pro-Gly-amide 34

Et pGlu-His-Trp-Ser-His-D-Nal-Trp-Arg-Pro-NHEt 35

Et pGlu-His-Trp-Ser-His-D-Nal-Leu-Tyr-Pro-NHEt 36

Et pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Tyr-Pro-NHEt 37

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-Gly-amide 38

CH₂CONH₂ pGlu-His-Trp-Ser-Try-D-Leu-Trp-Arg-Pro-Gly-amide 39

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-Gly-amide 40

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Leu-Trp-Arg-Pro-Gly-amide 41

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Leu-Leu-Tyr-Pro-Gly-amide 42

Et pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-NHEt 43

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Tyr-Pro-Gly-amide 44

Et pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Arg-Pro-NHEt 45

Et pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-NHEt 46

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-Gly-amide 47

Et pGlu-His-Trp-Ser-His-D-Leu-Trp-Arg-Pro-NHEt 48

Et pGlu-His-Trp-Ser-His-D-Leu-Leu-Tyr-Pro-NHEt 49

Et pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Tyr-Pro-NHEt 50

Et pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-NHEt 51

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-Gly-amide 52

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-amide 53

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-Gly-amide 54

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Bhi-Trp-Arg-Pro-Gly-amide 55

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Bhi-Leu-Try-Pro-Gly-amide 56

Et pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-NHEt 57

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-amide 58

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-amide 59

Et pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-NHEt 60

Et pGlu-His-Trp-Ser-His-D-Bhi-Trp-Arg-Pro-NHEt 61

Et pGlu-His-Trp-Ser-His-D-Bhi-Leu-Tyr-Pro-NHEt 62

Et pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Tyr-Pro-NHEt 63

Et pGlu-His-Trp-Ser-His-D-Bhi-Trp-Tyr-Pro-NHEt 64

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Tyr-Pro-Gly-amide 65

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Trp-Trp-Tyr-Pro-Gly-amide 66

CH₂CONH₂ pGlu-His-Trp-Ser-Tyr-D-Trp-Trp-Arg-Pro-Gly-amide 67

Et pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-NHEt 68

CH₂CONH₂ pGlu-His-Trp-Ser-His-D-Trp-Leu-Arg-Pro-Gly-amide

Of particular interest are compounds that are GnRH II analoguespredominantly having i) stimulating effect on or ii) affinity for typeII GnRH receptor. Thus, compounds are preferred that do not bind to oractivate type I GnRH receptor resulting in an undesired therapeuticresponse. It is contemplated that GnRH II analogues that do bind to oractivate type I GnRH receptor thereby stimulating endocrine signallingare administered together with a sex hormone to counter endocrineeffects.

The present invention also provides a method for treating intracellularinfections such as infections by intracellular bacteria, protozoa andfungi.

The compounds of the invention is contemplated to induce improved MHC IIand MHC I antigen presentation, which make them useful in the treatmentof intracellular bacterial, fungal, and protozoal infections. Thepresent invention further provides a method for treating intracellularinfection which comprises administration of an unphysiological amount ofGnRH or a GnRH analog.

In Case that GnRH Analogs of the Invention Interact with Type I GnRHReceptor

GnRH analogs are known in the art. A GnRH analog is an agent that mimicsthe action of GnRH on the receptors of the anterior pituitary gland whenadministered to an animal including man. Whereas administration of aGnRH analog in a single low physiological dose or in single lowphysiological doses spaced in time does stimulate the receptors of theanterior pituitary gland and thus acts as a receptor agonist, thecontinuous administration of a GnRH analog in a high unphysiologicaldose per time unit will, after initial stimulation of the receptors ofthe anterior pituitary gland, inhibit the secretion of FSH and LH, andthus acts as a receptor antagonist.

Inhibition of FSH and LH secretion induces pharmacological castration.In this context, an unphysiological dose of GnRH or GnRH analog is adose resulting in an unphysiological plasma level of GnRH or GnRH,respectively, which may result in a castrating plasma level. In thiscontext, an unphysiological plasma level of GnRH or GnRH analog is alevel not comprised by range of levels of GnRH normally present or, incase of GnRH analogs, a level not comprised in regard of physiologicaleffect by the normal physiological effect range of GnRH in a healthyperson.

More particularly, an unphysiological plasma level of GnRH is a levelincreased, in particular increased for an extended period of time suchas for more than a week or more than a month, in respect of the normalphysiological plasma level of GnRH. Also, more particularly, anunphysiological plasma level of GnRH analog is an increased plasma levelof GnRH analog not comprised in respect of physiological effect by thenormal physiological effect range GnRH in a healthy person, inparticular not for an extended period of time such as for more than aweek or more than a month. Any useful form of administration of the GnRHor GnRH analog of the invention including their pharmaceuticallyacceptable salts is comprised by the invention, in particularintravenous, intramuscular, subcutaneous, sublingual, and nasaladministration. Particular preferred are depot and slow or sustainedrelease compositions.

If a compound of the present invention interact with type I GnRHreceptor and an undesired effect may be obtained, such a compound may beadministered in combination with one or more natural, semi-synthetic orsynthetic sex hormones to counter the endocrine effects of GnRH II-likepeptides, e.g. testosterone or oestrogen depending on the hormonalstatus of the patient. In an adult male person the natural,semi-synthetic or synthetic sex hormone is testosterone or an agenthaving a corresponding hormonal effect. In an adult female person thenatural, semi-synthetic or synthetic sex hormone is oestradiol or anagent having a corresponding hormonal effect, in particular incombination with a progestogen. The latter is added to avoid thedevelopment of endometrial cancer in the female and to avoid vaginalbleedings. Hysterectomized women, however, do not benefit from theaddition of progestogen.

The combined administration of one or more natural, semi-synthetic orsynthetic sex hormones can be i) at the same time, i) the GnRH analogcan be administered earlier than the sex hormone, or iii) the GnRHanalog can be administered later than the sex hormone. Moreover, anddependent on the administration form used, the GnRH analog and/or thehormone may be administered more than one time such as eg in case ofadministration of the sex hormone via nasal spray, where administrationtypically is one or more times a day during one or more weeks.

The combined administration may extend, for instance, for over one ormore periods interrupted by administration-free periods, or theadministration can be continuous. A preferred administration period isfrom one to two weeks, in particular from 10 to 14 days. If a compoundof the present invention has an endocrine effect by activating GnRH I itis preferred that the administration of said compound substantiallyoverlaps the period of administration of one or more natural,semi-synthetic or synthetic sex hormones, such as by more than 50 percent, preferably by more than 85 per cent, even more preferred by morethan 90 or 95 per cent. The combined administration allows to protectthe person from serious endocrine side effects, such as decreasedlibido, hot flushes, increased perspiration, and increased heart rate.In an adult male person the natural, semi-synthetic or synthetic sexhormone administered to counter the endocrine effect of a compound ofthe invention is testosterone or an agent having a correspondinghormonal effect, in particular synthetic or semisynthetic agents thatmimic the hormonal effects of testosterone. Preferred agents comprisemethyltestosterone and stanozolol. In an adult female person thenatural, semi-synthetic or synthetic sex hormone administered to counterthe endocrine effect of a compound of the invention is an oestrogen suchas oestradiol or a semi-synthetic ester of oestradiol or anothersynthetic or semisynthetic oestrogen analog. Preferred oestrogen analogscomprise conjugated oestrogens, ethynylestradiol, and mestranol, as wellas non-steroidal oestrogens such as dinestrol and diethylstilbestrol. Ina female said oestrogen or oestrogen analog administration is in oneaspect preferably combined in combination with administration of aprogestogen, in particular progesterone, a progesterone derivative oranalog, such as hydroxyprogesterone caproate, medroxyprogesteroneacetate, noethisterone acetate, megestrol acetate, medrogestone andnorgestrel. The combined administration preferably overlaps by more than50 per cent, preferably by more than 85 per cent, even more preferred bymore than 90 or 95 per cent. It is preferred that the progestogen to beadministered in combination with the oestrogen, the semisynthetic esterof oestradiol or estriol or the synthetic or semisynthetic oestrogenanalog continuously or over periods of from about 10 to 14 days inintervals from about one to three months.

If necessary, a natural, semisynthetic, or synthetic sex hormone isadministered in combination with a type I GnRH receptor-activatingcompound of the invention, and optionally with a pharmaceuticallyacceptable carrier.

General Chemistry Methods

The skilled person will recognise that the compounds of the inventionmay be prepared, in known manner, in a variety of ways. The routes beloware merely illustrative of some methods that can be employed for thesynthesis of compounds of formula (I).

In general, synthetic methods for preparing compounds of the inventioncan be divided into two methods: liquid phase synthesis and solid phasesynthesis. Liquid phase peptide synthesis involves reagents reactingtogether in the solution phase. Disadvantages of this method includedifficulty in separating and purification of the products. Solid phasepeptide synthesis is more common and has numerous advantages includingconvenient isolation and purification and applicability to automation(Bodanszky et al, In Peptide Synthesis, John Wiley & Sons, 1976). Manypeptide synthetic resins have been developed to allow synthesis ofvarious peptides. These include chloromethyl and 2-chlorotitrylpolystyrene resins. Examples of patents disclosing methods for synthesisof short peptides include U.S. Pat. No. 5,602,231, EP0518655 and U.S.Pat. No. 6,879,289.

When a compound of the invention is prepared with a C-terminal secondaryamide, as in e.g. buserelin, then one method of preparing the compoundsis as follows and depicted in scheme I below. The peptide can beassembled on a solid support, typically 2-chlorotrityl polystyrene resinis used, but others will be apparent to one skilled in the art. Thefirst amino acid is loaded and then deprotected to reveal a reactiveamine group that is then used to couple onto the next amino acid. Thisin turn can be deprotected and coupled. After multiple rounds ofextension, the desired peptide sequence is obtained. The peptide is thencleaved from the resin by the action of TFA or similar reagents. Notethat when a tert-butyl side chain is required in the final compound itis important to keep the reaction time low enough such that this doesnot cleave entirely. Some tert-butyl will cleave but this can be removedin purification. Finally, the secondary amide is prepared by couplingthe deprotected peptide at the C-terminus with a selected primary amine.Coupling reactions typically utilise HBTU and DIPEA, though one skilledin the art will be able to identify other activators and bases that canbe used in combination to effect the amide bond formation.

When a compound of the invention is prepared with a C-terminal primaryamide, as in e.g. triptorelin, then one method of preparing thecompounds is as follows and depicted in scheme II below. The peptide canbe assembled on a solid support, typically Ramage resin is used, butothers will be apparent to one skilled in the art. The first amino acidis loaded and then deprotected to reveal a reactive amine group that isthen used to couple onto the next amino acid. This in turn can bedeprotected and coupled. After multiple rounds of extension, the desiredpeptide sequence is obtained. The peptide is then cleaved from the resinby the action of TFA or similar reagents. Coupling reactions typicallyutilise HBTU and DIPEA, though one skilled in the art will be able toidentify other activators and bases that can be used in combination toeffect the amide bond formation.

Compounds of formula (I) may be made by combining the methods describeabove and by other methods known to one skilled in the art.

General Use of the Compounds of the Invention

Compounds as described herein can be used in medicine, medical researchor in the manufacture of a composition for such use. Moreover, theinvention also relates to the compounds P1-P21 as described herein foruse in medicine, medical research or in the manufacture of a compositionfor such use. Accordingly, when in the following the term “compounds ofthe invention” is used in connection with medical use or pharmaceuticalcomposition, the term is intended also to include compounds P1-P21provided that these compounds have not been known for such a use.

In addition, the compounds are contemplated to show improved propertiesfor treatment of intracellular infection and related diseases, includingreduced binding to type I GnRH receptor as compared to type II GnRHreceptor.

The compounds of the invention are contemplated to be of use in thetreatment of intracellular bacterial, fungal, and protozoal infections,such as Mycobacterium tuberculosis, Mycobacteria causing atypicaldisease, Mycobacterium avium and M. intracellulare (also known asMycobacterium avium-intracellulare complex, or MAC), M. kansasii, M.marinum, M. fortuitum, M. gordinae, Mycoplasma pneumoniae, M.genitalium, M. hominis, Ureaplasma urealyticum, U. parvum, Chlamydophilapneumoniae, and Salmonella typhimurium, and in the treatment ofintracellular protozoal infections, such as Toxoplasma gondii,Plasmodium falciparum, P. vivax, Trypanosoma cruzi, Cryptosporidium, andLeishmania, and intracellular fungal infections such as Histoplasmacapsulatum, Cryptococcus neoformans, and Encephalitozoon cuniculi whenthese infections occur alone or in association with viral agents orviral diseases, or in association with other causes of primary orsecondary immunodeficiency. Causes of primary immunodeficiency includeinherited genetic deficiencies and somatic mutations, whereas secondaryimmunodeficiency may be caused by viral infections such as thosedescribed above, or by inheritable or non-inheritable conditions such asDiabetes mellitus, or malnutrition, or by agents such asimmunodepressants, drug abuse, or other environmental factors.

Moreover, the compounds of the invention disclosed herein may be used asa co-treatment to of viral diseases, disorders, conditions, andsymptoms, such as in treating patients infected with viral agents orwith viral diseases such as HIV, Adenovirus, Alphavirus, Arbovirus,Borna Disease, Bunyavirus, Calicivirus, Condyloma Acuminata,Coronavirus, Coxsackievirus, Cytomegalovirus, Dengue fever virus,Contageous Ecthyma, Epstein-Barr virus, Erythema Infectiosum,Hantavirus, Viral Hemorrhagic Fever, Viral Hepatitis, Herpes SimplexVirus, Herpes Zoster virus, Infectious Mononucleosis, Influenza, LassaFever virus, Measles, Mumps, Molluscum Contagiosum, Paramyxovirus,Phlebotomus fever, Polyoma-virus, Rift Valley Fever, Rubella, SlowDisease Virus, Smallpox, Subacute Sclerosing Panencephalitis, TumorVirus Infections, West Nile Virus, Yellow Fever Virus, Rabies Virus andRespiratory Syncitial Virus.

Moreover, the compounds are contemplated to be suitable for use in thetreatment or co-treatment of cancer. In particular, Adrenal Cancer, AnalCancer, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain/CNS Tumors,Breast Cancer, Castleman Disease, Cervical Cancer, Colon/Rectum Cancer,Endometrial Cancer, Esophagus Cancer, Eye Cancer, Gallbladder Cancer,Gastrointestinal Carcinoid Tumors, Gastrointestinal Stromal Tumor(GIST), Gestational Trophoblastic Disease, Hodgkin Disease, KaposiSarcoma, Kidney Cancer, Laryngeal and Hypopharyngeal Cancer, AcuteMyeloid Leukemia, Chronic Lymphocytic Leukemia, Acute LymphocyticLeukemia, Chronic Myeloid Leukemia, Chronic Myelomonocytic Leukemia,Liver Cancer, Non-Small Cell Lung Cancer, Small Cell Lung Cancer, LungCarcinoid Tumor, Lymphoma, Malignant Mesothelioma, Multiple Myeloma,Myelodysplastic Syndrome, Nasal Cavity and Paranasal Sinus Cancer,Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Oral Cavityand Oropharyngeal Cancer, Osteosarcoma, Ovarian Cancer, PancreaticCancer, Penile Cancer, Pituitary Tumors, Prostate Cancer,Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Basal andSquamous Cell Skin Cancer, Melanoma, Merkel Cell Skin Cancer, SmallIntestine Cancer, Stomach Cancer, Testicular Cancer, Thymus Cancer,Thyroid Cancer, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer,Waldenstrom Macroglobulinemia, Wilms Tumor.

Thus the advantageous properties of the compound of the invention mayinclude one or more of the following:

-   -   Improved binding to type II GnRH receptor as compared to type I        GnRH receptor    -   Improved MHC class I stimulation    -   improved MHC class II stimulation    -   Improved immunomodulation    -   Improved activation of antigen presenting cells    -   Improved T-cell response    -   Improved antiviral activity    -   Improved anticancer activity    -   Improved MHC II antigen presentation    -   Improved MHC I antigen presentation

Pharmaceutical Compositions Comprising a Compound of the Invention

The present invention also provides a pharmaceutical compositioncomprising the com-pound of the invention together with one or morepharmaceutically acceptable diluents or carriers. The present chapter isprimarily directed to formulation of the novel GnRH analogs. In thosecases where the novel compounds have an effect on type I GnRH receptor,which is unwanted and causes castration or similar effects, compositionscontaining sex hormones are known in the art and may be co-administered.

The compounds of the invention or a formulation thereof may beadministered by any conventional route for example but withoutlimitation it may be administered parenterally, orally, topically or viaa mucosa (including buccal, sublingual, transdermal, vaginal, rectal,nasal, ocular etc.), via a medical device (e.g. a stent), by inhalation.The treatment may consist of a single administration or a plurality ofadministrations over a period of time.

The treatment may be by administration once daily, twice daily, threetimes daily, four times daily etc. dependent on the specific disease tobe treated and the weight and age of the patient to be treated. Thetreatment may also be by continuous administration such as e.g.administration intravenous by infusion via a drop.

Whilst it is possible for the compound of the invention to beadministered as such, it is preferable to present it as a pharmaceuticalformulation, together with one or more acceptable carriers. Thecarrier(s) must be “acceptable” in the sense of being compatible withthe compound of the invention and not deleterious to the recipientsthereof. Examples of suitable carriers are described in more detailbelow.

The formulations may conveniently be presented in a suitable dosage formincluding a unit dosage form and may be prepared by any of the methodswell known in the art of pharmacy. Such methods include the step ofbringing into association the active ingredient (compound of theinvention) with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

The compound of the invention will normally be administered by anyconventional administration route normally by the oral or any parenteralroute, in the form of a pharmaceutical formulation comprising the activeingredient, optionally in the form of a nontoxic organic, or inorganic,acid, or base, addition salt, in a pharmaceutically acceptable dosageform. Depending upon the disorder and patient to be treated, as well asthe route of administration, the compositions may be administered atvarying doses and/or frequencies.

The pharmaceutical compositions must be stable under the conditions ofmanufacture and storage; thus, if necessary should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.In case of liquid formulations such as solutions, dispersion, emulsionsand suspensions, the carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (e.g. glycerol,propylene glycol and liquid polyethylene glycol), vegetable oils, andsuitable mixtures thereof.

For example, the compound of the invention may be administered orally,buccally or sublingually in the form of tablets, capsules, films,ovules, elixirs, solutions, emulsions or suspensions, which may containflavouring or colouring agents.

Formulations in accordance with the present invention suitable for oraladministration may be presented as discrete units such as capsules,cachets or tablets, each containing a predetermined amount of the activeingredient; as multiple units e.g. in the form of a tablet or capsule:as a powder or granules; as a solution or a suspension in an aqueousliquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion ora water-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

Solutions or suspensions of the compound of the invention suitable fororal administration may also contain one or more solvents includingwater, alcohol, polyol etc.as well as one or more excipients such aspH-adjusting agent, stabilizing agents, surfactants, solubilizers,dispersing agents, preservatives, flavors etc. Specific examples includee.g. N,N-dimethylacetamide, dispersants e.g. polysorbate 80,surfactants, and solubilisers, e.g. polyethylene glycol, Phosal 50 PG(which consists of phosphatidylcholine, soya-fatty acids, ethanol,mono/diglycerides, propylene glycol and ascorbyl palmitate). Theformulations according to present invention may also be in the form ofemulsions, wherein a compound according to Formula (I) may be present inan emulsion such as an oil-in-water emulsion or a water-in-oil emulsion.The oil may be a natural or synthetic oil or any oil-like substance suchas e.g. soy bean oil or safflower oil or combinations thereof.

Tablets may contain excipients such as microcrystalline cellulose,lactose (e.g. lactose monohydrate or lactose anyhydrous), sodiumcitrate, calcium carbonate, dibasic calcium phosphate and glycine,butylated hydroxytoluene (E321), crospovidone, hypromellose,disintegrants such as starch (preferably corn, potato or tapiocastarch), sodium starch glycollate, croscarmellose sodium, and certaincomplex silicates, and granulation binders such as polyvinylpyrrolidone,hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC),macrogol 8000, sucrose, gelatin and acacia. Additionally, lubricatingagents such as magnesium stearate, stearic acid, glyceryl behenate andtalc may be included.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder (e.g. povidone, gelatin, hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (e.g. sodium starchglycolate, crosslinked povidone, cross-linked sodium carboxymethylcellulose), surface-active or dispersing agent. Moulded tablets may bemade by moulding in a suitable machine a mixture of the powderedcompound moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provideslow or controlled release of the active ingredient therein using, forexample, hydroxypropylmethylcellulose in varying proportions to providedesired release profile.

Solid compositions of a similar type may also be employed as fillers ingelatin capsules. Preferred excipients in this regard include lactose,starch, a cellulose, milk sugar or high molecular weight polyethyleneglycols. For aqueous suspensions and/or elixirs, the compounds of theinvention may be combined with various sweetening or flavouring agents,colouring matter or dyes, with emulsifying and/or suspending agents andwith diluents such as water, ethanol, propylene glycol and glycerin, andcombinations thereof.

Formulations suitable for topical administration in the mouth includelozenges comprising the active ingredient in a flavoured basis, usuallysucrose and acacia or tragacanth; pastilles comprising the activeingredient in an inert basis such as gelatin and glycerin, or sucroseand acacia; and mouth-washes comprising the active ingredient in asuitable liquid carrier.

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, impregnated dressings, sprays, aerosols oroils, transdermal devices, dusting powders, and the like. Thesecompositions may be prepared via conventional methods containing theactive agent. Thus, they may also comprise compatible conventionalcarriers and additives, such as preservatives, solvents to assist drugpenetration, emollient in creams or ointments and ethanol or oleylalcohol for lotions. Such carriers may be present as from about 1% up toabout 98% of the composition. More usually they will form up to about80% of the composition. As an illustration only, a cream or ointment isprepared by mixing sufficient quantities of hydrophilic material andwater, containing from about 5-10% by weight of the compound, insufficient quantities to produce a cream or ointment having the desiredconsistency.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active agent may be delivered from the patch byiontophoresis.

For applications to external tissues, for example the mouth and skin,the compositions are preferably applied as a topical ointment or cream.When formulated in an ointment, the active agent may be employed witheither a paraffinic or a water-miscible ointment base.

Alternatively, the active agent may be formulated in a cream with anoil-in-water cream base or a water-in-oil base.

For parenteral administration, fluid unit dosage forms are preparedutilizing the active ingredient and a sterile vehicle, for example butwithout limitation water, alcohols, polyols, glycerine and vegetableoils, water being preferred. The active ingredient, depending on thevehicle and concentration used, can be either colloidal, suspended ordissolved in the vehicle. In preparing solutions the active ingredientcan be dissolved in water for injection and filter sterilised beforefilling into a suitable vial or ampoule and sealing.

Advantageously, agents such as local anaesthetics, preservatives andbuffering agents can be dissolved in the vehicle. To enhance thestability, the composition can be frozen after filling into the vial andthe water removed under vacuum. The dry lyophilized powder is thensealed in the vial and an accompanying vial of water for injection maybe supplied to reconstitute the liquid prior to use.

Pharmaceutical compositions of the present invention suitable forinjectable use include sterile aqueous solutions or dispersions.Furthermore, the compositions can be in the form of sterile powders forthe extemporaneous preparation of such sterile injectable solutions ordispersions. In all cases, the final injectable form must be sterile andmust be effectively fluid for easy syringability.

Parenteral suspensions are prepared in substantially the same manner assolutions, except that the active ingredient is suspended in the vehicleinstead of being dissolved and sterilization cannot be accomplished byfiltration. The active ingredient can be sterilised by exposure toethylene oxide before suspending in the sterile vehicle. Advantageously,a surfactant or wetting agent is included in the composition tofacilitate uniform distribution of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents. A person skilled in the art will know how tochoose a suitable formulation and how to prepare it (see eg Remington'sPharmaceutical Sciences 18 Ed. or later). A person skilled in the artwill also know how to choose a suitable administration route and dosage.

It will be recognized by one of skill in the art that the optimalquantity and spacing of individual dosages of a compound of theinvention will be determined by the nature and extent of the conditionbeing treated, the form, route and site of administration, and the ageand condition of the particular subject being treated, and that aphysician will ultimately determine appropriate dosages to be used. Thisdosage may be repeated as often as appropriate. If side effects developthe amount and/or frequency of the dosage can be altered or reduced, inaccordance with normal clinical practice.

A pharmaceutical composition may also be a two-part composition, whereone part contains the GnRH analogue and the other part contains the sexhormone. The two parts may be combined eg as a two-layer tablet or theymay be present eg as pellets in a capsule. Known compositions containinga GnRH analogue and known compositions containing a sex hormone may alsobe used in a method of the invention.

All % values mentioned herein are % w/w unless the context requiresotherwise.

Sequence List

The sequence list is prepared according to the WIPO standard ST.25. Inthe sequence list, the unnatural amino acids of compounds 1-63 andP1-P21 are represented as the corresponding natural amino acid in thefollowing way:

Corresponding natural amino Unnatural amino acid acid pGlu,pyroglutamate L-Glutamate, Glu

L-Serine, Ser

L-Tryptophan, Trp

L-Phenylalanine, Phe

L-Histidine, His

L-Leucine, Leu Pro-Et L-Proline, Pro Pro-NHCONH₂ L-Proline, Pro Gly-NH₂Gly

In the sequence list, entries 1-63 correspond to compounds 1-63, andentries 64-78 correspond to compounds P1-P15. Entries 79 and 80correspond to wild-type GnRH I and GnRH II. Entries 81-84 correspond toprimers. Entries 85-89 correspond to compounds 64-68. Entries 90-95correspond to compounds P16-P21. However, the sequences SEQ ID Nos: 1-78and 85-89 as they are stated in the sequence list, i.e. withoutabove-described unnatural amino acids, are not claimed, but are includedonly to comply with the requirements of R. 30(1) of the EPC.

Repetition of Free Text from Sequence Listing

For compliance with paragraph 36 of WI PO Standard ST.25, the free textincluded under numeric identifier <223> of the sequence listing ishereby repeated in the main part of the description:

SEQ ID NO Free text included in <223>  1-78 Man-made analogue of GnRH II79 GnRH I 80 GnRH II 81 Type I GnRH Receptor forward primer 82 Type IGnRH Receptor reverse primer 83 Type II GnRH Receptor forward primer 84Type II GnRH Receptor reverse primer 85-95 Man-made analogue of GnRH II

Definitions

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. at least one) of the grammatical objects of the article.By way of example “an analogue” means one analogue or more than oneanalogue.

As used herein the terms “immunorhelins” and “compound(s) of theinvention” are used interchangeably and refer to compounds of formula(I).

The pharmaceutically acceptable salts of the compound of the inventioninclude conventional salts formed from pharmaceutically acceptableinorganic or organic acids or bases as well as quaternary ammonium acidaddition salts. More specific examples of suitable acid salts includehydrochloric, hydrobromic, sulfuric, phosphoric, nitric, perchloric,fumaric, acetic, propionic, succinic, glycolic, formic, lactic, maleic,tartaric, citric, palmoic, malonic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, toluenesulfonic, methanesulfonic,naphthalene-2-sulfonic, benzenesulfonic hydroxynaphthoic, hydroiodic,malic, steroic, tannic and the like. Other acids such as oxalic, whilenot in themselves pharmaceutically acceptable, may be useful in thepreparation of salts useful as intermediates in obtaining the compoundsof the invention and their pharmaceutically acceptable salts. Morespecific examples of suitable basic salts include sodium, lithium,potassium, magnesium, aluminium, calcium, zinc,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, N-methylglucamine and procaine salts.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : Expression of MHC class I after stimulation of T cells withincreasing concentrations of GnRH II. PBMCs from a healthy donor wasstimulated with GnRH II and IL-2 for 72 hours. Data points representmean fluorescent intensity of MCH class I expression on CD4⁺ T cells(blue triangles) or CD8⁺ T cells (black squares) measured with flowcytometry.

FIGS. 2A-2B: Expression of MHC class I after stimulation of T cells withincreasing concentrations of GnRH I analogue (red) and GnRH II. (black).PBMCs from a healthy donor was stimulated with GnRH I analogoue or withGnRH II and IL-2 for 72 hours. Data points represent mean fluorescentintensity of MCH class I expression on CD4⁺ T cells (FIG. 2A) or CD8⁺ Tcells (FIG. 2B) measured with flow cytometry.

FIG. 3 : Expression of MHC class I after stimulation of CD4⁺CD14⁺monocytes with increasing concentrations of GnRH I analogue (red) andGnRH II (black). CD14⁺ monocytes PBMCs from a healthy donor wasstimulated with GnRH I analogue or with GnRH II and IL-2 for 72 hours.Data points represent mean fluorescent intensity of MCH class Iexpression on CD4⁺CD14⁺ monocytes measured with flow cytometry.

FIG. 4 : GnRH receptor expression in human T cells analysed withquantitative real-time PCR. The bars represent ratios of GnRHR I orGnRHR II mRNA normalized to RNA polymerase II expression in sorted naiveT cells (white bars) or memory T cells (gray bars). MCF-7 breast cancercell line (black bar) was used as a positive control.

FIG. 5 : Expected data: Number of mycobacteria in the lungs of infectedmice are suppressed by compounds of the present invention in thepresence or absence of testosterone. Colony Forming Units (CFU) areassessed by growing lysates of lung tissue from infected mice onbacterial plates and counting colonies.

EXPERIMENTAL General Biology Methods

The preferential effect of the compounds of the invention on GnRHreceptors may be tested using one or more of the methods describedbelow:

I. Expression of GnRH Receptors on T Cells

Human naive and memory T cells were labeled with fluorescent surfacemarker antibodies CD45RA, CD45RO and CD4 and sorted with flow cytometry.Total RNA was extracted with Rnaeasy kit (Qiagen) and reversedtranscribed with iScript select cDNA synthesis kit (Biorad). Thetemplate cDNA was amplified with SYBR Green (Applied Biosystem) and runon CFX96 PCR (Biorad). Ratios of Type I GnRH Receptor and Type II GnRHReceptor mRNA were normalized to RNA polymerase II expression in sortednaive T cells or memory T cells. The MCF-7 breast cancer cell line wasused as a positive control.

Type I GnRH Receptor fwd 5′-tgc ctc ttc atc atc cct ct-3′ rev5′-gca aat gca acc gtc att tt-3′ Type II GnRH Receptor fwd5′-act gtt caa tgg ctg gct gt-3′ rev 5′-gcc ccc aga agt ttc ctt ac-3′

I. GnRH I vs GnRH II Assay

Compounds were tested on cells made to express Type I or Type II GnRHReceptors by transfection. The cells were exposed to labelled GnRHcompound, washed and then assessed by measuring the label on the cells.The label was either measured directly (radioactive isotope label orfluorescent label) or indirectly (biotin labelled peptide).

Signalling induced by the GnRH compounds was measured in the cell linesexpressing Type I GnRH and Type II GnRH Receptors respectively. GnRHcompounds were investigated for their respective affinity to type I GnRHand type II GnRH receptors using competition assays. Calcium flux wasmeasured using cells labelled with Fluo-4-Direct either using a flowcytometer or by live cell imaging microscopy, in order to evaluate theirpotency establishing ED50 values. Signalling was also studied by westernblotting using antibodies to p-ERK or p-JNK.

To assess the effects of cellular activation on the production of LH andFSH and compare it with stimulation of immune related functions, theeffects of the compounds were studied on pituitary cells and immunecells expressing either Type II GnRH or Type I GnRH Receptors.

II. Expression of Cell Specific Surface Markers and MHC Class II and MHCClass I

Human peripheral blood mononuclear cells (PBMCs) were purified fromhealthy donors with Ficoll-Hypaque density centrifugation. Cells werecultured in RPMI-1640 medium (Invitrogen) supplemented with 10% fetalbovine serum, 100 μg/mL ampicilin and 100 μg/mL streptomycin for 24-72hours in 37° C., 5% CO₂ Cells were stimulated with a compound accordingto the invention and analysed for expression of cell specific surfacemarkers and MHC class II (monoclonal antibodies from BD Pharmingen) withflow cytomtery.

To test a set of compounds according to the invention for theirimmunomodulatory properties in an in vitro assay and evaluate theirability to induce MHC class II expression on monocytes. First when aknown GnRH analogue was used in a co-culture to stimulate monocytes. Asmall increase in MHC class expression from background of in MFI may beseen. In contrast, when a compound according to the invention is used wemay detect a larger expression of cell surface expression of MHC classII. If this is the observation, we may have identified a compound withan effect on MHC class II expression, allowing increased turnover andpresentation of MHC class II and class I peptides from the endosomal andlysosomal pathway. The findings will enhance the presentation ofpeptides derived from intracellular pathogens and promote CD4⁺ helper Tas well as CD8+ T cells cells activation, expansion and inducesterilizing immunity.

Materials

Unless otherwise indicated, all reagents used in the examples below areobtained from commercial sources.

Theoretical Example of the Compounds of the Invention on IntracellularBacteria Material and Methods

Male mice are infected with mycobacterium tuberculosis by inhalation ofan aerosol containing the bacteria. The infecting dose is between 100and 1000 bacteria per mouse. The GnRHII or GnRHI related compound (aloneor together with testosterone) or vehicle is administered by anappropriate route in an appropriate dose for 1-2 weeks followinginfection with the bacteria either. The mice are sacrificed and lungsremoved and homogenized and plated on bacteria dishes containing mediumthat supports the growth of mycobacterium tuberculosis. After 3-4 weeksincubation, in a heated cabinet at 37 degrees C., the amount of bacteriain the lungs of the mice are quantified by counting bacterial colonieson the plate.

Expected Results

Expected results of this experiments is that the amount of mycobacteriumtuberculosis bacteria in the lungs of mice are reduced in mice treatedwith GnRH compounds compaired with control vehicle treated mice. We alsoexpect GnRHII related compounds to be superior to GnRHI relatedcompounds in this respect. We do not expect co-administration oftestosterone will have any effect on the degree of effect of the GnRHcompounds.

Measurement of Castractive Effects and Compensation Thereof

Castration induced by the compounds of the invention, as well as anycompensation thereof can be determined by measurement of the circulatinglevels of the relevant sex hormones. How to carry out such measurementsis known to the person skilled in the art.

General Synthesis Method

Peptides were prepared using standard Fmoc solid-phase synthesis as perthe diagram above. Protected amino acids (Fmoc and tBu or Trt ifnecessary) were used, and synthesis was performed on 2-chlorotritylpolystyrene resin. Reactions are carried out in the order A, B, Cfollowed by multiple iterations of B and C to build up the desiredpeptide. When the final amino acid (pyroglutamate—note, reaction C isused to do this, though the amino acid is not Fmoc protected) have beenadded the final two reactions—D and E—take place in that order togenerate a compound of the invention.

Reaction A: The resin was suspended in dichloromethane (10-20 volumeequivalents compared to the resin) and stirred at room temperature. Fmocprotected amino acid (2 equivalents) was added to 1 equivalent of resinin the presence of of diisopropylethylamine (6 equivalents). Thereaction was stirred for 0.5 to 1 hour at room temperature. The resinwas collected by filtration and washed 6 times with DMF and then useddirectly in the next step.

Reaction B: The Fmoc protecting group was removed by the treatment ofpiperidine (20%) in dimethylformamide (5-10 volume equivalents comparedto the resin) at room temperature. The reaction was stirred for up to 1hour and the resin collected b filtration and then the resin was washed6 times with DMF and used directly in the next step.

Reaction C: Fmoc-protected amino acid (4 equivalents) was dissolved inDMF and DIPEA (2 equivalents) added. After stirring at room temperaturefor one minute these were added to the resin supported amino acid (1equivalent) from Reaction B was treated with HBTU (1 equivalent) added.The reaction was stirred for up to one hour and before the resin wascollected by filtration and washed 6 times with DMF and used directly inthe next step. The next step was either reaction B or reaction Ddepending on the target sequence.

Reaction D: The protected peptide was cleaved from the resin bytreatment with 3-5% trifluoroacetic acid in dichloromethane. The resinwas removed by filtration and the peptide accrued by precipitation withice cold diethyl ether and collection by centrifugation. The solid waswashed in further diethyl ether and then dried under vacuum before beingused in the next step.

Reaction E: The C-terminal amide was formed by dissolving the peptidefrom Reaction D (1 equivalent) in DMF, monoalkylamine (20-50equivalents) and HBTU (2-3 equivalents) were added and the reactionstirred at room temperature for up to 3 hours. The reaction was dilutedwith water and the crude peptide was then purified as detailed below.

Peptides were prepared using standard Fmoc solid-phase synthesis as perthe diagram above. Protected amino acids (Fmoc and tBu or Trt ifnecessary) were used, and synthesis was performed on Ramage resin.Reactions are carried out in the order A, B, C followed by multipleiterations of B and C to build up the desired peptide. When the finalamino acid (pyroglutamate—note, reaction C is used to do this, thoughthe amino acid is not Fmoc protected) have been added the final tworeactions—D and E—take place in that order to generate a compound of theinvention.

Reaction F: Fmoc Ramage resin is suspended in DMF (5-10 volumeequivalents compared to resin) containing 20% piperidine. The reactionwas stirred for up to 1 hour at room temperature and the resin collectedby filtration and washed 6 times with DMF and used directly in the nextreaction.

Reaction G: Fmoc-protected amino acid (5 equivalents) was dissolved inDMF and DIPEA (2 equivalents) added. After stirring at room temperaturefor one minute these were added to the resin supported amino acid (1equivalent) from Reaction F was treated with HBTU (1 equivalent) added.The reaction was stirred for up to one hour and before the resin wascollected by filtration and washed 6 times with DMF and used directly inthe next step.

Reaction H: The Fmoc protecting group was removed by the treatment ofpiperidine (20%) in dimethylformamide (5-10 volume equivalents comparedto the resin) at room temperature. The reaction was stirred for up to 1hour and the resin collected b filtration and then the resin was washed6 times with DMF and used directly in the next step.

Reaction I: Fmoc-protected amino acid (4 equivalents) was dissolved inDMF and DIPEA (2 equivalents) added. After stirring at room temperaturefor one minute these were added to the resin supported amino acid (1equivalent) from Reaction H was treated with HBTU (1 equivalent) added.The reaction was stirred for up to one hour and before the resin wascollected by filtration and washed 6 times with DMF and used directly inthe next step. The next step was either reaction H or reaction Jdepending on the target sequence.

Reaction J: The peptide was cleaved from the resin by treatment with 90%trifluoroacetic acid with 2.5% water, 2.5% triisopropylsilane and 5%dichloromethane. The resin was removed by filtration and the peptideaccrued by precipitation with ice cold diethyl ether and collection bycentrifugation. The crude peptide was then purified as detailed below.

Purification

The crude peptides were individually dissolved in acetonitrile/H₂O (1:1,v/v) and purified by preparative HPLC with a C18 column using a water(0.1% TFA)-acetonitrile (0.1% TFA) gradient. The final purity of thepeptides was confirmed by analytical HPLC. Peptide was lyophilizedbefore storage at −20° C.

Compound Analysis—Identity and Purity

Analysis Method A

For analysis, the compounds were dissolved in methanol:water (9:1, 0.1mg/ml) and a 150 μl portion was placed in an HPLC microvial andcentrifuged at 14000 rpm for 3 minutes. The sample was then examined byhigh performance liquid chromatography with diode array (HPLC-DAD) andmass spectrometry (HPLC-MS) detection. HPLCDAD-MS was performed using anAgilent 1100 HPLC system comprising of quaternary pump, auto sampler,column oven and diode array detector coupled to a Waters ZQ singlequadrupole mass spectrometer. The same reverse-phase Waters Xselect CSHC18, 2.1 mm×50 mm, 3.5 μm particle size column was used for allcompounds and was fitted with a Waters VanGuard CSH C18, 2.1 mm×5 mm,3.5 μm particle size guard column and Waters Acquity, 0.2 μm in-linecolumn filter. The column was used at a flow rate of 1 ml/min maintainedat a temperature of 60° C. The solvents used were % formic acid in 95%acetonitrile, 5% water (solvent B) and 10 mM ammonium formate, 0.2%formic acid in water (solvent A), with a gradient as follows: 5% solventB from 0 to 0.2 min, 5 to 50% solvent B from 0.2 to 9.3 min, 50 to 95%solvent B from 9.3 to 9.5 min, 95% solvent B from 9.5 to 11 min, 95 to5% solvent B from 11 to 11.05 min and re-equilibration with 5% solvent Bfrom 11.05 to 11.5 min. Nitrogen was used as auxiliary and sheath gas.Source voltage was set at 3400 V, cone voltage set at 31 V with a gasflow of 50 L/hour, drying gas flow rate at 550 L/hour and drying gastemperature at 350° C.

Compound Analysis—Solubility and Stability in Solution

Analysis Method B

For solubility and stability analysis, the compounds were dissolved (0.2mg/ml) in phosphate buffer solution (PBS, 10 mM, pH 7.4) and shaken atroom temperature for 20 minutes. A T=0 hour sample was taken (80 μl) andcentrifuged at 14000 rpm for 3 minutes then analysed by Analysis methodA as above. The bulk samples were placed in a Techne Roller-Blot HB-3DRolling Hybridiser at 37° C. and only removed when a sample (80 μl) wastaken at time points T=4, 24 and 96 hours. The samples were centrifugedat 14000 rpm for 3 mins then analysed by HPLC-DAD-MS as above. The UVarea under curve at 280 nm was recorded at each time point.

EXAMPLES Example 1—Compound Synthesis

Compounds of the invention were made according to the methods set out inthe General Synthesis Method.

Com- pound Synthesis no. R1 R2 R3 R4 R5 Method  6

Et A  8

Et A  9

Et A 10

Et A 11

Et A 12

Et A 13

Et A 14

CH₂CONH₂ B 16

CH₂CONH₂ B 20

CH₂CONH₂ B 23

CH₂CONH₂ B 24

CH₂CONH₂ B 25

CH₂CONH₂ B 27

CH₂CONH₂ B 28

CH₂CONH₂ B 30

CH₂CONH₂ B 33

CH₂CONH₂ B 42

Et A 44

Et A 45

Et A 47

Et A 48

Et A 49

Et A 50

Et A 56

Et A 59

Et A 60

Et A 61

Et A 62

Et A 63

Et A 64

CH₂CONH₂ 65

CH₂CONH₂ 66

CH₂CONH₂ 67

Et 68

CH₂CONH₂

Compound Retention Time m/z (Analysis number Salt form (Analysis methodA) method A) 6 TFA 4.15 1213.8 8 TFA 5.5 1312.7 9 TFA 6.64 1246.8 10 TFA4.27 1286.9 11 TFA 5.35 1220.9 12 TFA 6.75 1319.7 13 TFA 5.53 1293.7 14TFA 6.13 1318.7 16 TFA 4.93 1292.8 20 TFA 4.02 1358.9 23 TFA 4.79 1296.724 TFA 5.84 1395.7 25 TFA 7.19 1329.6 27 TFA 4.91 1369.6 28 TFA 5.921303.8 30 TFA 7.20 1402.7 33 TFA 6.00 1376.4 42 TFA 3.90 1183.8 44 TFA5.23 1282.8 45 TFA 6.35 1216.8 47 TFA 4.00 1256.8 48 TFA 5.12 1190.8 49TFA 6.56 1290.0 50 TFA 5.30 1263.7 56 TFA 3.42 1297.7 59 TFA 5.22 1330.860 TFA 3.81 1370.6 61 TFA 4.36 1304.7 62 TFA 5.60 1403.5 63 TFA 4.691377.7 64 TFA 6.19 1391.7 65 TFA 5.09 1366.0 66 TFA 4.92 1384.7 67 TFA4.47 1396.9 68 TFA 3.83 1285.8

Example 2—Solubility Analysis

The solubility of compounds of the invention was tested as described inthe general methods. Solubility was then graded according to a ratingbetween 1 to 5, where 1 is most soluble and 5 is least soluble.

Compound number Solubility grading Buserelin acetate 1 Triptorelinacetate 2 Naferelin acetate 2 Histrelin acetate 4 Leuprorelin acetate 2Buserelin TFA 2 Triptorelin TFA 2 Naferelin TFA 1 Histrelin TFA 2Leuprorelin TFA 1  6 1  8 2  9 1 10 1 11 1 12 4 13 3 14 4 16 2 20 3 23 124 5 25 4 33 2 30 1 28 2 27 1 56 1 67 1 59 4 63 5 62 5 61 3 60 2 42 1 442 45 2 50 1 49 4 48 2 47 1 68 1 66 5 14 4 65 4 64 5 16 2 20 3

Example 3—Stability Analysis

The stability of compounds of the invention in aqueous media (PBS ph7.4)was tested as described in the general methods. Stability was thengraded according to a rating where t1/2>96 minutes was shown as + andstability less than this was shown as −.

Compound number Stability grading  6 +  8 +  9 + 10 + 11 + 12 + 13 +14 + 16 + 20 + 23 + 24 − 25 − 33 − 30 − 28 + 27 + 56 + 67 − 59 − 63 − 62− 61 + 60 + 42 + 44 + 45 + 50 + 49 + 48 + 47 + 68 + 66 + 14 + 65 + 64 −16 + 20 +

Example 4—GnRH R Stimulation

The ability of compounds of the invention to stimulate GnRHR wasassessed by using a calcium assay with CHO-K1 cells (Genscript), see thegeneral methods for details.

Activity was then recorded as percentage stimulation at 1 μM.

Compound number GNRHR Stimulation at 1 μM Buserelin  94 Leuprorelin 107(n = 2) Goserelin  99 Gonadorelin 102 Nafarelin 100 (n = 2)  6  88  8 86  9  78 10  81 11  76 12  80 13  87 14  78 16  76 20  75 23 106 24 87 25  82 33  67 30  12 28  95 27 108 56  96 67 117 59  85 63  77 62 77 61 112 60 102 42 102 44 111 45 105 50 105 49  77 48 118 47 119 68 86 66  83 14  78 65  72 64  50 16  76 20  75

REFERENCES

Interferon-inducible effector mechanisms in cellautonomous immunity.MacMicking, J. D. Nat. Rev. Immunol. 12,367-382 (2012).

Toll-like receptor 2-dependent inhibition of macrophage class II MHCexpression and antigen processing by 19-kDa lipoprotein of Mycobacteriumtuberculosis. Noss, E. H. et al. J. Immunol. 167,910-918 (2001).

HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 andClathrin but Not PACS-1 and Is Impeded by AP-2. Lubben, N. B. et al. MolBiol. Cell. 18 (3351-3365).

Processing of Mycobacterium tuberculosis antigen 85B involvesintraphagosomal formation of peptide-major histocompatibility complex IIcomplexes and is inhibited by live bacilli that decrease phagosomematuration. Ramachandra, L., Noss, E., Boom, W. H. & Harding, C. V. J.Exp. Med. 194,1421-1432 (2001).

The ins and outs of MHC class II-mediated antigen processing andpresentation. Paul A. Roche & Kazuyuki Furuta. Nature Reviews Immunology15, 203-216 (2015)

Secreted Toxoplasma gondii molecules interfere with expression of MHC-IIin interferon gamma-activated macrophages. Leroux L -P, Dasanayake D,Rommerein L M, Fox B A, Bzik D J, Jardim A, Dzierszinski F S.International Journal for Parasitology 2015 45: 319-332

Protection form Direct Cerebral Cryptococcus Infection by Interferongamma-dependent Activation of Microglial Cells. Zhou Q, Gault R A, KozelT R, Murphy W J. The Journal of Immunology 2007: 178: 5753-5761.

Mycobacterium tuberculosis EsxH inhibits ESCRT-dependent CD4+ T-cellactivation Portal-Celhay C, Tufariello J A M, Srivastava S, Zahra A,Klevorn T, Grace P S, Mehra A, Park H S, Ernst J D, Jacobs Jr W R &Philips J A. Nature Microbiology 2, Article number: 16232 (2016)doi:10.1038/nmicrobio1.2016.232

Gonadotropin secretion and its control. Fink G, The physiology ofreproduction 1998.

Immunomodulatory actions of gonadal steroids may be mediated bygonadotropin-releasing hormone, Jacobson J D and Ansari M A,Endocrinology 2004:145(1):330-6.

Unusual morphologic features of uterine leiomyornas treated withgonadotropin-releasing hormoneagonists: massive lymphoid infiltrationand vasculitis. McClean G and McCluggage W G, Int J Surg Pathol.2003;11(4):339-44.

Massive lymphocytic infiltration of uterine leyomyomas associated withGnRH agonist treatment. Bardsley V et al., Histopathology1998;33(1):80-2.

Chronic plasma cell endometritis in hysterectomy specimens ofHIV-infected women: a retrospective analysis. Kerr-Layton J A et al.,Infect Dis Obstet Gynecol. 1998;6(4):186-90.

Serum dihydrotestosterone and testosterone concentrations in humanimmunodeficiency virus-infected men with and without weight loss. ArverS et al., J Androl 1999:20(5):611-8.

Prevalence of endocrine dysfunction in HIV-infected men. Brookmeyer G etal., Horm Res 2000;54(5-6):294-5.

Gonadotropin-releasing hormone increases CD4-T-lymphocyte numbers in ananimal model of immunodeficiency. Jacobson J D et al., J Allergy ClinImmunol. 1999;104:653-8.

A transcriptionally active human type II gonadotropin-releasing hormonereceptor gene homolog overlaps two genes in the antisense orientation onchromosome 1q.12. Morgan et al., Endocrinology. 2003February;144(2):423-36

Gonadotropin-releasing hormone (GnRH)-binding sites in human breastcancer cell lines and inhibitory effects of GnRH antagonists. Eidne etal., J Clin Endocrinol Metab. 1987 March;64(3):425-32

All references referred to in this application, including patent andpatent applications, are incorporated herein by reference to the fullestextent possible.

1-21. (canceled)
 22. A method for treating an intracellular infection ina human or animal subject, the method comprising administering atherapeutically effective amount of a GnRH analog according to formula(I):

or a pharmaceutically acceptable salt thereof to the subject in needthereof; wherein R₁=

 R₂=

 R₃=

 R₄=

and  R₅═Me, Et, CH₂CF₃, iPr, nPr, nBu, iBu, sBu, tBu, cyclopropyl,CH₂CONH₂, or NHCONH₂; and wherein one of R₁, R₃, and R₄ is selected fromType I according to the list below, and two of R₁, R₃, and R₄ areselected from Type II according to the list below: Group Type I Type IIR₁

R₃

R₄


23. The method according to claim 22, wherein the intracellularinfection is selected from an intracellular bacterial, an intracellularfungal, and an intracellular protozoal infection.
 24. The methodaccording to claim 22, wherein the intracellular infection is selectedfrom Mycobacterium tuberculosis, Mycobacteria causing atypical disease,Mycobacterium avium and M. intracellulare (also known as Mycobacteriumavium-intracellulare complex, or MAC), M. kansasii, M. marinum, M.fortuitum, M. gordinae, Mycoplasma pneumoniae, M. genitalium, M.hominis, Ureaplasma urealyticum, U. parvum, Chlamydophila pneumoniae,Salmonella typhimurium, Toxoplasma gondii, Plasmodium falciparum, P.vivax, Trypanosoma cruzi, Cryptosporidium, Leishmania, Histoplasmacapsulatum, Cryptococcus neoformans, and Encephalitozoon cuniculi. 25.The method according to claim 22, wherein R₅=Et or CH₂CONH₂.
 26. Themethod according to claim 22, wherein R₅═Me, iPr, nPr, nBu, iBu, sBu, ortBu.
 27. The method according to claim 22, wherein R₁=


28. The method according to claim 27, wherein: R₂=


29. The method according to claim 27, wherein: R₁=

R₃=

and R₄=


30. The method according to claim 27, wherein the GnRH analog accordingto formula (I) is one of the following comuounds: Compound no. R₁ R₂ R₃R₄ R₅ 28

CH₂CONH₂ 35

Et 55

CH₂CONH₂ 61

Et 41

CH₂CONH₂ or 48

Et

or a pharmaceutically acceptable salt of any of these.
 31. The methodaccording to claim 27, wherein: R₁=

R₃=

and R₄=


32. The method according to claim 31, wherein the GnRH analog accordingto formula (I) is one of the following compounds: Compound no. R₁ R₂ R₃R₄ R₅ 27

CH₂CONH₂ 34

Et 54

CH₂CONH₂ 60

Et 40

CH₂CONH₂ or 47

Et

or a pharmaceutically acceptable salt of any of these.
 33. The methodaccording to claim 27, wherein R₂=


34. The method according to claim 33, wherein the GnRH analog accordingto formula (I) is one of the following compounds: Compound no. R₁ R₂ R₃R₄ R₅ 5

CH₂CONH₂ 11

Et 4

CH₂CONH₂ or 10

Et

or a pharmaceutically acceptable salt of any of these.
 35. The methodaccording to claim 22, wherein: R₁=

R₃=

and R₄=


36. The method according to claim 35, wherein the GnRH analog accordingto formula (I) is one of the following compounds: Compound no. R₁ R₂ R₃R₄ R₅ 7

CH₂CONH₂ 12

Et 30

CH₂CONH₂ 36

Et 62

Et 43

CH₂CONH₂ or 49

Et

or a pharmaceutically acceptable salt of any of these.
 37. A method fortreating an intracellular infection in a human or animal subject, themethod comprising administering a therapeutically effective amount of aGnRH analog or a pharmaceutically acceptable salt thereof to the subjectin need thereof, wherein the GnRH analog is one of the followingcompounds: 1: pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-Gly-NH₂, 2:pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-Gly-NH₂, 3:pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-Gly-NH₂, 6:pGlu-His-Trp-Ser-His-D-Ser(tBu)-Leu-Arg-Pro-NHEt, 8:pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Trp-Arg-Pro-NHEt, 9:pGlu-His-Trp-Ser-Tyr-D-Ser(tBu)-Leu-Tyr-Pro-NHEt, 23:pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-Gly-NH₂, 24:pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-Gly-NH₂, 25:pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-Gly-NH₂, 26:pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Arg-Pro-NHEt, 29:pGlu-His-Trp-Ser-His-D-Nal-Leu-Arg-Pro-NHEt, 31:pGlu-His-Trp-Ser-Tyr-D-Nal-Trp-Arg-Pro-NHEt, 32:pGlu-His-Trp-Ser-Tyr-D-Nal-Leu-Tyr-Pro-NHEt 33:pGlu-His-Trp-Ser-His-D-Nal-Trp-Tyr-Pro-Gly-NH₂, 37:pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-Gly-NH₂, 38:pGlu-His-Trp-Ser-Try-D-Leu-Trp-Arg-Pro-Gly-NH₂, 39:pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-Gly-NH₂, 42:pGlu-His-Trp-Ser-His-D-Leu-Leu-Arg-Pro-NHEt, 44:pGlu-His-Trp-Ser-Tyr-D-Leu-Trp-Arg-Pro-NHEt, 45:pGlu-His-Trp-Ser-Tyr-D-Leu-Leu-Tyr-Pro-NHEt, 50:pGlu-His-Trp-Ser-His-D-Leu-Trp-Tyr-Pro-NHEt, 51:pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-Gly-NH₂, 52:pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH₂, 53:pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-Gly-NH₂, 56:pGlu-His-Trp-Ser-His-D-Bhi-Leu-Arg-Pro-NHEt, 57:pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH₂, 58:pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-Gly-NH₂, 59:pGlu-His-Trp-Ser-Tyr-D-Bhi-Leu-Tyr-Pro-NHEt, 63:pGlu-His-Trp-Ser-His-D-Bhi-Trp-Tyr-Pro-NHEt, or 67:pGlu-His-Trp-Ser-Tyr-D-Bhi-Trp-Arg-Pro-NHEt.
 38. The method according toclaim 37, wherein the intracellular infection is selected from anintracellular bacterial, an intracellular fungal, and an intracellularprotozoal infection,
 39. The method according to claim 37, wherein theintracellular infection is selected from Mycobacterium tuberculosis,Mycobacteria causing atypical disease, Mycobacterium avium and M.intracellulare (also known as Mycobacterium avium-intracellularecomplex, or MAC), M. kansasii, M. marinum, M. fortuitum, M. gordinae,Mycoplasma pneumoniae, M. genitalium, M. hominis, Ureaplasmaurealyticum, U. parvum, Chlamydophila pneumoniae, Salmonellatyphimurium, Toxoplasma gondii, Plasmodium falciparum, P. vivax,Trypanosoma cruzi, Cryptosporidium, Leishmania, Histoplasma capsulatum,Cryptococcus neoformans, and Encephalitozoon cuniculi.